HIV, the causative agent of AIDS, is a complex retrovirus that encodes several genes in addition to the structural genes commonly found in murine retroviruses. The functions of two of these genes, tat and rev, have been largely elucidated on a molecular basis, by virtue of the clear phenotypes associated with the Tat and Rev proteins. In essence, the products of both genes are required for virus replication, and removal of either gene results in failure to replicate in virtually any known system. In contrast, the vif, vpr, vpu, and nef genes are generally not required for viral replication in different in vitro systems. In vivo, however, they all act together as important determinants of virulence, although their individual contributions are not always obvious. Even in animal models that simulate the complex pattern of pathology associated with AIDS, the roles of some of these genes are unclear. Nef, for example, which has been shown to virtually have an all-or-nothing effect in the development of an AIDS-like syndrome in adult macaques, is not required to induce disease in neonates. Considerable evidence indicates that the levels of HIV/SIV replication largely determine the final outcome of the disease and that inhibition of virus replication by any means results in significant benefits to infected patients. We hypothesize that inhibition of Nef function can significantly reduce virus replication in vivo and that such inhibition will positively affect the final outcome of infection. Even if these benefits do not extend to neonates, the majority of infected adults are likely to benefit from Nef inhibitors. The focus of this R21 grant is the regulation of Nef function by posttranslational modifications, specifically the role of phosphorylation on Nef function. Because the rational development of Nef inhibitors requires a deeper understanding of how Nef function is regulated, we propose the following specific aims: 1) To identify the phosphorylated residues of Nef. 2) To determine the role of phosphorylation on Nef activities. To accomplish these aims we have recruited three outstanding collaborators with extensive expertise in state-of-the-art proteomics and classic biochemical approaches that together with our track record on Nef will provide important information regarding the identity of the phophorylation sites on Nef and their functional role. [unreadable] [unreadable] [unreadable]